Connexin-43 is a promising target for lycopene preventing phthalate-induced spermatogenic disorders

Graphical abstract

The present study identified a critical role of Cx43 in LYC maintaining BTB integrity, which is a prerequisite for normal spermatogenesis. This study reveals the role of Cx43 in LYC preventing phthalate-induced spermatogenic disorders, and Cx43 regulation may provide the basis for future therapeutic male infertility.

Highlights

• •Phthalate induced spermatogenic disorders and destroyed BTB integrity.
• •Phthalate caused cell migration capacity decline and cytoskeletal disorganization.
• •LYC alleviated DEHP-induced disruption of intercellular junctions in mouse testes.
• •LYC improved DEHP-induced spermatogenic disorders by regulating BTB integrity.
• •LYC ameliorated DEHP-induced BTB disruption by modulating Cx43 expression.

Abstract

Introduction

Male infertility is a multifactorial pathological condition and may be a harbinger of future health. Phthalates are ubiquitous environmental contaminants that have been implicated in the global decline in male fertility. Among them, di-(2-ethylhexyl) phthalate (DEHP) is the most prevalently used. Lycopene (LYC) is a possible preventive and therapeutic agent for male infertility owing to its antioxidant properties. The blood-testis barrier (BTB) is formed between Sertoli cells where it creates a unique microenvironment for spermatogenesis.

Objectives

We hypothesize that phthalate caused male infertility and LYC plays an important role in phthalate-induced male fertility disorders.

Methods

Hematoxylin-eosin (H&E) staining, ultrastructure observation, and fluorescence microscopy were used to examine the morphological changes. RNA-Seq, and western blotting were conducted to detect gene and protein levels. Routine testing for sperm morphology and sperm-egg binding assay were conducted to examine the morphological structure and function of sperm. Cell scratch assay and transepithelial electrical resistance (TER) were used to detect cell migration capacity and barrier integrity.

Results

In vivo experiments, we showed that LYC prevented DEHP-induced impairment of BTB integrity, which provided a guarantee for the smooth progress of spermatogenesis. LYC improved DEHP-induced change in sperm parameters and fertilization ability. Subsequent in vitro experiments, LYC alleviated MEHP-induced disruption of intercellular junctions in mouse Spermatogonia cells (GC-1 cells) and mouse Sertoli cells (TM4 cells). In MEHP-induced BTB impairment models of Sertoli cells, treatment with LYC or overexpressing connexin-43 (Cx43) promoted cell migration capacity and normalized BTB integrity. Cx43 knockdown inhibited cell migration capacity and perturbed BTB reassembly in LYC preventing DEHP-induced BTB impairment.

Conclusion

Our study provides evidence for the role of LYC in phthalates-induced spermatogenic disorders and points to Cx43 as a potential target for male fertility.

Introduction

Phthalates are ubiquitous environmental contaminants that impair male reproduction in rodents via their endocrine-disrupting properties [1]. More than 470 million pounds of phthalates are imported and produced annually, accounting for 65 % of global plasticizer consumption [2]. Overall global phthalates consumption grows at an average annual rate of 1.3 % between 2017 and 2022 [3]. Di-(2-ethylhexyl) phthalate (DEHP) is one kind of the most common phthalates and universally used in polyvinyl chloride (PVC) products. The US Environmental Protection Agency (EPA) had considered DEHP as a priority pollutant [4]. It has been estimated that the average daily exposure of DEHP is 3 and 30 μg/kg/day, which is the reference dose set by the U.S. EPA [5]. Once in the body, DEHP is metabolized by several enzymes into metabolites with MEHP as the primary metabolite, and the MEHP were commonly detected in human liver, blood, urine and reproductive organs [6]. Thereby, DEHP is labeled as endocrine disrupting chemicals (EDCs) in both male and female reproductive systems, causing diverse diseases including reproductive and metabolic diseases [7], [8]. While ample male reproductive toxicity studies of DEHP have been reported, it is little understanding of the potential molecular drivers of DEHP-induced male infertility.

Lycopene (LYC) is one of the natural powerful antioxidant agents owing to thirteen conjugated double bonds in the molecular structure [9]. It has been also shown that the antioxidant capacity of LYC is 47 times as of β-carotene and 100 times as of vitamin E respectively [10], [11]. Furthermore, evidences in human and animal studies have not reported any side effects of LYC [12]. The main sources of LYC are tomato-based products, which exceed 80 % of food source for human. LYC mainly accumulates in the testis, liver and prostate, and the concentration in the testes is as high as ten times than in other tissues, which may be owing to presence of many lipoprotein uptake, or higher metabolic/oxidation [13]. The association between LYC and male reproductive disease has been studied for more than two decades [14]. For this purpose, it is necessary to investigate whether LYC could be applied to the treatment of DEHP-induced male reproductive diseases.

Male factor infertility accounts for half of all infertility cases, being only found in 30 % of them, and no cause can be identified in 70 % of the male of infertile couples [15]. Throughout spermatogenesis in the human testis, germ cells develop into spermatozoa through consecutive cell divisions and differentiation. Spermatogenesis is entirely dependent on blood-testis barrier (BTB), which separates advanced germ cells in the testis from the immune system that otherwise be considered “foreign”[16]. Although a partial loss of BTB structure and function results in male infertility, during sperm production the BTB structure briefly ‘open’ and ‘close’ to allow initial germ cells to enter the immune microenvironment [17]. Several cellular junctions (tight junctions (TJ), adherent junction (AJ), and gap junction (GJ)) function to establish BTB comprised of diverse structural proteins. Simultaneously, the three components act synergistically in BTB to allow germ cell maturation to proceed [18]. However, the extent to which DEHP-induced BTB disruption affects male fertility and the therapeutic effects of LYC on it are poorly understood.

Here, we show a direct correlation between LYC and the connexin-43 (Cx43) protein. Using a range of mouse models coupled to three distinct vitro models of mouse testicular cells, we showed that LYC prevented DEHP-induced spermatogenesis disorders by maintaining the structure and function of BTB in mouse testis. We further link gap junction function to interaction at the BTB and the spermatogenesis. What’s more, we proved that LYC-mediated restoration of Cx43 protein alleviated DEHP-induced decreased cell migration capacity, the disorder and decrease of cytoskeleton morphological structure and BTB destruction. Finally, we showed that BTB integrity can be stabilized as a result of LYC-mediated increase in Cx43 level. As GJ regulates the local tissue microenvironment, we hypothesize that GJ stabilization, via Cx43 targeting, represents a unique way of maintaining the structure and function of BTB, especially in treatment resistant male infertility patients.

Materials and methods

Ethics statement

All procedures for animal experiments were conducted in the Guidelines for Care and Use of Laboratory Animals of Northeast Agricultural University (NEAU). Experiments were approved by the Animal Ethics Committee of Northeast Agricultural University (NEAUEC20220341). All animal experiments should comply with the ARRIVE guidelines.

Mice

The ICR male mice were obtained from Liaoning Changsheng Biotech Co., ltd., (Liaoning, China). The experimental mice were 3-week of age and housed under controlled temperature, humidity, and light (12 h light on/off cycle) with food and water readily available ad libitum (Table S1). During the experiment, the body weight, food intake and water consumption were measured daily. After four weeks of daily gavages, the mice were sacrificed by cervical dislocation and the collected tissues were frozen at −80 °C until used. DEHP and LYC were purchased from Aladdin Biochemical Technology Co., ltd., (Shanghai, China) and Solaibao Biological Technology Co., ltd. (Beijing, China), respectively. The doses given to animals are selected primarily in reference to previous studies [19], [20], [21].

Hematoxylin-eosin (H&E) staining

Dissected testes were fixed in 4 % paraformaldehyde, embedded in paraffin, and cut into 4 μm thick sections. The sections were then deparaffinized, stained with H&E and viewed and photographed using a digital scanner microscope (Panoramic MIDI, 3DHISTECH ltd., Hungary).

RNA-seq

RNA-seq analysis for testicular tissue (n = 3) and TM4 cells (n = 3) were executed by Shenggong Bioengineering Co., ltd (Shanghai, China) and Shanghai WeiHuan Biotech Co., ltd (Shanghai, China), respectively. RNA extraction, library construction and RNA-seq data analysis were carried out in RNA-seq analysis following the previous method [22]. The detailed methods are presented in Supplementary 1.1.

Ultrastructure observation

Electron microscopy of tissues and cells were performed at the Electron Microscope Center of Northeast Agricultural University. Briefly, the tissues and cells were fixed with glutaraldehyde, postfixed in OsO4 and then stained. The images were captured using a transmission electron microscope (HT7650, Hitachi, Japan).

Analysis of BTB integrity

Fluorescein isothiocyanate (FITC) method was performed to assess BTB integrity with minor modification as previous study [23]. Briefly, the mice were anesthetized and FITC were administered via intravenous tail vein injections. After 60 min, the mice were euthanized by cervical dislocation. The testis of mice was placed in OCT embedding medium, which was cut into frozen sections. Finally, the distribution of FITC in the testes was observed using fluorescence microscope (Leica, Germany). The mice were treated with CdCl₂ (3 mg/kg) by intraperitoneal injection before the last day of the experiment as positive control.

Routine testing for sperm morphology

The sperm was collected from cauda epididymides and placed in physiological saline (37 °C). The sperm was stained by Quick sperm stain kit (Nanjing Jiancheng Bioengineering Institute Co., ltd., Nanjing, China). The head, mid-piece and tail of sperm were observed with an EVOS imaging system (Invitrogen, USA).

Sperm-egg binding assay

The female ICR mice (8-week-old) were injected intraperitoneally with pregnant mare serum gonadotrophin (PMSG), then injected with human chorionic gonadotrophin (hCG) 48 h later. About 14  h after hCG injection, egg was collected from the ampulla of the oviduct and placed in 200 μL Toyoda-Yokoyama-Hosoki (TYH) medium under mineral oil. First, the sperm was placed in TYH containing hoechst and incubated for 10 min at 37 °C with 5 % CO₂. Then, dispersed cultured sperm (20 μL) were removed into TYH medium (200 μL), and incubate for 50 min to enable sperm capacitation of fresh cauda epididymis in male mice. Finally, an aliquot (10 μL) of the capacitated sperm was mixed with the above TYH medium containing eggs, and incubated for 2 h. Finally, the sperm-egg binding was observed using fluorescence microscope (Leica, Germany).

Western blot

To determine the proteins level change, the total protein was extracted from samples using RIPA buffer with protease/phosphatase inhibitor cocktail (MCE, USA). Briefly, total proteins were separated using SDS-PAGE and transferred to nitrocellulose (NC) membranes, which were then blocked with 5 % nonfat milk. The membranes were incubated with primary antibodies (Abcam, England; CST, USA; Affinity, USA; Proteintech, USA; ABclonal, Wuhan, China; Bioss, Beijing, China), and then incubated with secondary antibodies (Zhongshan Jinqiao Biotechnology Co., ltd., Beijing, China). After washing with TBST, the protein membranes were visualized by using Amersham Imager (GE, Switzerland).

Immunofluorescence (IF) analyses

Testes tissues of mice were embedded and sectioned (5 μm-thick). Then, we performed IF staining of mouse testicular tissue for E-cadherin, N-cadherin, Occludin, ZO-1, Cx43 and Vimentin as described in detail in Supplementary 1.2. The images of IF were observed using fluorescence microscope (Leica, Germany).

In vitro evaluation

GC-1, TM3 and TM4 cell lines (ATCC, USA) were cultured in DMEM (high glucose and F12, respectively) (HyClone, USA) with FBS (10 %) (Biological Industries, Israel), penicillin–streptomycin (1 %) (Gibco, USA) at 37 °C with 5 % CO₂. The cells were cultured every 1–2 days to maintain cells in the growth logarithmic phase. Firstly, LYC (Sigma-Aldrich) was dissolved in Tetrahydrofuran (THF) containing 0.025 % butylated hydroxytoluene (BHT) and incubated in cells for 4 h. Then, MEHP (AccuStandard Inc., USA) was dissolved in DMSO and incubated in cells for 24 h. The DMSO or THF amount in culture medium did not exceed 0.1 % (v/v), a concentration that did not affect the assays (results were similar to application of vehicle-free control medium). The doses given to cells are selected primarily in reference to previous studies [24], [25].

Cell viability analysis

The cell viabilities were measured by the CCK8 assay. The cells were seeded in 96-well plates at 1 × 10⁴ cells per well. After different treatments with DEHP and/or LYC, the cells were incubated with CCK8 reagent for 1 h. The absorbance was read at 450 nm using a microplate reader (Shanghai, China).

AO/EB staining assay

AO/EB double staining kit (Shanghai China) was used in this experiment. After treatment, the cells were digested using 0.25 % pancreatin and stained with AO/EB working fluid. Finally, the AO/EB staining was observed using a fluorescence microscope (Leica, Germany).

Cell IF

The cells were fixed with 4 % paraformaldehyde and permeabilized with 0.1 % Triton X-100 for 15 min. After washing with PBS, the cells were incubated in 1 % BSA and 10 % FBS for 1 h, incubated with diluted primary antibodies (Abcam, England; CST, USA; Affinity, USA; Proteintech, USA; ABclonal, Wuhan, China; Bioss, Beijing, China) overnight at four degrees, washed with PBST, and incubated with Alex 488/596‐conjugated secondary antibodies (Abcam, England) for 60 min at 37 °C. The cells were washed with PBS and then stained with DAPI. The cell IF was observed using fluorescence microscope (Leica, Germany).

Cell scratch assay

The cells were seeded in a 6-well culture plate and allowed to reach confluence. A scratch was made using a gun head (200 μL). After treatment, the images were captured with an EVOS imaging system (Invitrogen, USA) and the scratch wound area was assessed at 0, 12, 24 and 48 h, respectively.

Evaluation of cell morphology

Fluorescence staining of the microfilament proteins (F-actin) and microtubule proteins (α-tubulin) was performed by double staining. After treatment, the cells were fixed with 4 % paraformaldehyde and permeabilized using 0.1 % Triton X-100. The cells were stained using F-actin (Shanghai, China) and α-tubulin (Nanjing, China) for 30 min, respectively, and further stained with DAPI (Nanjing, China) for 10 min. the evaluation of cell morphology was observed using fluorescence microscope (Leica, Germany).

Isolation and treatment of primary Sertoli cells (SCs)

Mouse SCs were prepared as described previously and described in detail in Supplementary Methods 1.3. Furthermore, SCs were identified by WT-1 staining. According to the previous studies [26], LYC (1 μM) and/or MEHP (200 μM) were added at SC cells and cultured for 4 h and/or 24 h.

Measurement of transepithelial electrical resistance (TER)

TER is widely used to evaluate cellular barrier functions and also a classical method commonly used in the study of BTB integrity. We quantify the TER with a Millicell ERS system (Billerica, MA). Measurement of TER were described in detail in Supplementary Methods 1.4. Briefly, SCs were seeded on transwell chamber and cultured to allow BTB assembly. After treatment, TER reading was recorded daily. Medium without cells was used as a blank control. The TER was calculated through the formula: TER (Ω cm²) = (treatment resistance (Ω)-background resistance(Ω)) × membrane area (cm²).

Cell transfection

Before transfection, TM4 cells and SCs were seeded and cultured in DMEM/F12 medium with 10 % FBS (without dual-antibody). When the TM4 cells and SCs confluency was almost 50 %-70 %, the DNA constructs and siRNAs were transfected into cells by Lipofectamine 3000 (Invitrogen, USA) for 24 h. The expression vector, used for overexpression of Cx43, was purchased from Shenggong Bioengineering Co., ltd (Shanghai, China). siRNA duplexes were obtained from Ruibo Biotechnology Co., ltd (RiboBio, China), as shown below: si-Cx43 (target sequence: CCGCAATTACAACAAGCAA). The whole pcDNA3.1 plasmid and negative control siRNA (siNC) were used as negative controls.

Statistics

GraphPad Prism 8.0 software was used to perform all statistical analyses. All in vitro and vivo experiments were carried out with a minimum of three parallel samples. Values are expressed as mean ± SD. One-way ANOVA and Tukey’s post-test multiple comparisons were used to compare data with different groups. P ≤ 0.05 were considered significant.

Results

LYC prevents DEHP-induced disruption of BTB integrity in mice

In the seminiferous epithelium of testis, the BTB located above the basement membrane is critical to the homeostasis in the testis and the process of sperm production (Fig. 1A). Earlier studies have already confirmed that phthalate exposure destroy BTB integrity of testes, and thus impair male fertility [27]. Since we have recently showed that the potential men’s health supplement, LYC, is a feasible therapeutical option for male infertility [28], we decide to test whether LYC may improve phthalate-induced spermatogenesis disturbance by regulating BTB integrity. BTB, constituted between Sertoli cells in the seminiferous tubules, is a crucial ultrastructure in the mammalian testis. Therefore, ultrastructure is a common method to observe BTB structure. The typical “Sandwich” structure at the BTB appeared intact in Con, Vcon and LYC groups. As expected, the vesicles, disintegration and fragmentation at the BTB were observed in DEHP exposure group, but this disruption was reversed by LYC treatment (Fig. 1B and S1E). To further explore if LYC prevents DEHP-induced disruption of BTB integrity, we used a FITC assay. In the Con, Vcon and LYC testis, FITC signals only were observed in the lateral basement membrane of seminiferous tubules, but not in the seminiferous tubule lumen. As expected, the FITC signals penetrated seminiferous tubules in DEHP-exposed group, but LYC treatment suppressed this signal penetration (Fig. 1D).

Fig. 1.

LYC prevents DEHP-induced disruption of BTB integrity in mice. (A) BTB creates a distinct microenvironment for spermatogenesis and sperm maturation. (B) Changes of BTB structure in mice after exposure to water (Con), corn oil (Vcon), LYC (5 mg/kg LYC), D500 (500 mg/kg DEHP) and DL500 (5 mg/kg LYC + 500 mg/kg DEHP). (C) The levels of AJ, TJ, GJ and Vimentin proteins in mouse testicular tissue. (D) The function and integrity BTB in mouse testis. Scale bars, 200 μm. (E) Expression and positioning of E-cadherin and N-cadherin proteins in mouse testicular tissue. Scale bars, 100 μm. (F) Expression and positioning of ZO-1 and occludin proteins in mouse testicular tissue. Scale bars, 100 μm. (G) Expression and positioning of Cx43 protein in mouse testicular tissue. Scale bars, 100 μm. (H) Expression and positioning of Vimentin protein in mouse testicular tissue. Scale bars, 100 μm.

The BTB is created by the related proteins of TJs, AJs and GJs that are constructed between Sertoli cells [29]. Next, we assessed the expression BTB-related proteins at different treatment of DEHP and/or LYC. DEHP-exposed mice showed decreased levels of AJ proteins (Integrin β1, N-cadherin, E-cadherin, α-catenin, β-catenin, γ-catenin), GJ proteins (Cx43), TJ proteins (Claudin5, Claudin 11, ZO-1, Occludin) and vimentin. However, LYC treatment improved most of these parameters (Fig. 1C and S2A-B). Besides, the impacts of LYC on DEHP-induced changes of BTB-related proteins are further verified using IF staining. The result suggested the presence of N-cadherin, ZO-1, Occludin, Cx43 and Vimentin in the Sertoli cells, and E-cadherin in the Leydig cells (Fig. 1E-H). As displayed in Fig. 1E, the N-cadherin, E-cadherin, ZO-1, Occludin, Cx43 and Vimentin are obviously reduced in DEHP-treated testes and the locations of these proteins were not different, but LYC treatment suppressed these signal decrease. Taken together, these findings highlight that LYC plays a protective role in DEHP-induced impairment of BTB integrity.

LYC improves DEHP-induced spermatogenic disorders by regulating BTB integrity

We and others first demonstrated that the potential men’s health supplement, LYC, could improve sperm parameters and male infertility [30], and thus we decide to test whether LYC may improve DEHP-induced spermatogenesis disturbance by regulating BTB integrity. First, the mice showed signs of reduced feed intake and no significant difference in the consumption of water (Fig. S1A-C). We found that the structures of the seminiferous tubules in the DEHP-treated groups were disrupted, loosened, disordered, or unclear. DEHP treatment decreased the number of spermatogenic cell layers and mature sperm per luminal cross-section (Fig. S1D). Spermatids undergo maturation via spermiogenesis to form spermatozoa which are to be released into the tubule lumen at spermiation. In our study, GO enrichment analysis showed that DEHP caused mitochondrial impairment and increased ROS generation, which could disrupt sperm motility and viability [31]. Consistently, GO enrichment analysis also showed that spermatid development, spermatid differentiation and spermatogenesis were significantly enriched after exposure to DEHP (Fig. 2A-B and S6D). Furthermore, germ cell development, cellular process involved in reproduction, male gamete generation and sexual reproduction were also obviously enriched after DEHP exposure, suggesting that DEHP could impact negatively on male reproductive outcomes, especially sperm damage. Heatmap of differentially expressed genes shows the GO enrichment analysis of spermatogenesis-related genes (Fig. 2C), which showed that DEHP can affect the testicular growth and immune function, and thereby might destroy the immune-privileged environment for spermatogenesis. The data are in agreement with previous study, proving that DEHP could cause testicular dysfunction and interfere with the spermatogenesis process. To confirm the role of LYC in DEHP-induced spermatogenesis disorder, we further perform the test of sperm structure and function. DEHP exposure caused the breakdown and disappearance of nuclear membrane and mitochondrial cristae in spermatogonia. Furthermore, the nuclear malformation, change of acrosome structure, and cytoplasmic residues in sperm head were shown in DEHP-treated group. In the DL500 group, the nucleus and mitochondrial structures of spermatogonia were normal, the cytoplasmic vacuoles in spermatogonia were reduced, and the sperm heads and acrosomes also returned to normal (Fig. 2D). The integrity of sperm is critical for normal sperm function, and the abnormality of sperm consistently leads to male infertility [32]. Abnormal sperm morphology was observed in DEHP-exposed group, such as head deformities, folded tails and flagellar kinked. Meanwhile, the sperm malformation, TZI and SDI were also increased after treatment of DEHP compared with the Vcon group. However, the percentage of sperm malformation and malformed types were obvious reduced after LYC treatment (Fig. 2E). To evaluate the sperm fertilizing ability, we perform the spermatozoa penetrating the cumulus cells analysis. The results showed that the cumulus cell layer was not completely dissociated in the DEHP treatment group, and a large number of unseparated cumulus granulosa cells still adhered to the surface of the egg, which prevents the sperm from advancing. However, the cumulus cell layer was almost completely dissociated, and only part of the unseparated cumulus granulosa adhered to the egg surface in the DL500 group, which promotes the sperm from advancing (Fig. 2F). Overall, the data proves that LYC improves DEHP-induced spermatogenesis disturbance by regulating BTB integrity.

Fig. 2.

LYC improves DEHP-induced spermatogenesis disturbance. (A) The enrichment GO term after D500 exposure in mouse testicular tissue. (B) The enrichment dot bubble of GO (BP) analysis after D500 exposure in mouse testicular tissue. (C) Heatmap presentation of differential genes in spermatogenesis on mouse testis after exposure to D500. (D) Changes of ultrastructure of spermatogonium and sperm in mouse testicular tissue. (E) Papanicolaou staining of mouse sperm. (F) Changes in the function of the sperm penetrating the cumulus cell layers in mouse. Data are presented as the mean ± SD. Symbol for the significance of differences between the Vcon group and another group: *P < 0.05, ***P < 0.001. Symbol for the significance of differences between the D500 group and DL500 group: ^##P < 0.01, ^###P < 0.001.

LYC alleviates DEHP-induced disruption of intercellular junctions in GC-1 cells and TM3 cells

The BTB is constituted by TJ, GJ and AJ. The most notable AJ is ES (the testis-specific AJ), which is found in the Sertoli cell-spermatid cell and Sertoli cell-Sertoli cell interfaces [33]. Therefore, cell junction proteins on spermatogenic cells are also essential for the integrity of the intratesticular BTB. MEHP is the primary metabolite of the common plasticizing agent DEHP and considered to be more toxic than DEHP. We used CCK8 assay to establish the optimal concentration of the MEHP. When the MEHP concentration was 200 μM and 400 μM, the cell viability was near 80 % and 60 %, respectively. M400 (400 μM MEHP) significantly inhibited the cell viability of cells. Furthermore, the dosing of the MEHP was also set on the basis of previous studies [24] and preliminary experiments. To confirm the role of LYC on DEHP-induced cell junction proteins on spermatogenic cells, we selected LYC (1 μM) and/or MEHP (200 μM) for our subsequent experiments in GC-1 cells (Fig. 3A-B). After exposure to MEHP, the fracture of mitochondrial cristae, disappeared membranes, vacuoles, and a large number of autophagic vacuoles were shown in GC-1 cells. The ultrastructural structure of GC-1 cells in LYC treatment group returned to normal (Fig. 3C). LYC treatment also reduced DEHP-induced apoptosis and ROS production (Fig. 3D and S3D). We then tested the cell junction proteins expression by Western blot in GC-1 cells. As expected, LYC significantly rescued DEHP-induced the decrease of cell junction proteins expression, including AJ proteins (N-cadherin, E-cadherin, α-catenin, β-catenin, γ-catenin), GJ proteins (Cx43), TJ proteins (Claudin 5, ZO-1, Occludin) and Vimentin (Fig. 3E and S3A-B).

Fig. 3.

LYC alleviated DEHP-induced disruption of intercellular junctions in GC-1 cells. (A) GC-1 cells were incubated with Con (control), LYC (1 μM LYC), MEHP (200 μM MEHP) and ML200 (1 μM LYC + 200 μM MEHP), respectively. (B) The cell viability was assayed using CCK-8 after exposure to different concentrations of MEHP or LYC in GC-1 cells. (C) Transmission electron microscopy of the GC-1 cells; yellow: mitochondria. (D) The apoptosis and intracellular ROS production in GC-1 cells. (E) The levels of AJ, TJ, GJ and Vimentin proteins in GC-1 cells. (F) Changes of cellular migration ability in GC-1 cells. (G) Changes of cytoskeleton organization in GC-1 cells. Data are presented as the mean ± SD. Symbol for the significance of differences between the Con group and another group: *P < 0.05, **P < 0.01, ***P < 0.001.

Cell migration is an important functional property of cells and vital for normal development, immune regulation and disease processes. During cell migration, cells form anterior protrusions and new adhesions, which also play an important prerequisite for maintaining BTB integrity [34]. In our study, the influence of LYC and/or DEHP on the migration of GC-1 cells was investigated by scratch test. The mobility of DEHP dealing with GC-1 cells was significantly decreased compared with the Con group. However, LYC reversed DEHP-induced the reduced migration ability of GC-1 cell (Fig. 3F and S3C). Cell migration is a highly dynamic process mediated by the cytoskeleton (F-actin and a-tubulin) [35]. To verify whether DEHP and/or LYC treatment changes cell migration ability by regulating the cytoskeleton, we perform evaluation of cell morphology by double fluorescence staining of the microfilament proteins (F-actin) and microtubule proteins (α-tubulin). After exposure to DEHP, the expression of cytoskeletal components (F-actin and a-tubulin) was reduced and localization remained the same. However, LYC increased DEHP-induced decrease expression of cytoskeletal components, suggesting that LYC could promote cell-to-cell communication in GC-1 cells (Fig. 3G and S4A-B).

The building blocks of testicular intercellular junctions are now considered as major molecular regulators of male fertility [36]. The interactions of Leydig cells via intercellular junctions also provide the best environment for maintaining a normal testicular function. Next, we also selected LYC (1 μM) and/or MEHP (200 μM) for our subsequent experiments in TM3 cells. In agreement with the above results, LYC remarkly reversed DEHP-induced decrease of AJ proteins (N-cadherin, E-cadherin, α-catenin, β-catenin, γ-catenin), GJ proteins (Cx43), TJ proteins (Claudin 11, ZO-1, Occludin) and Vimentin (Fig. S5A-B). Meanwhile, LYC alleviated DEHP-induced decrease of migration ability and expression of cytoskeletal components (F-actin and a-tubulin) (Fig. S5C-F). Above all, our results document that LYC prevents DEHP-induced disruption of intercellular junctions in GC-1 cells and TM3 cells.

LYC prevented DEHP-induced disruption of continuous cell junctions in TM4 cells

Several protein complexes involved in BTB exert their regulatory effects downstream and BTB is mainly constituted with adjacent Sertoli cells [37]. To confirm the role of LYC in DEHP-induced cell junction proteins on Sertoli cell, we selected LYC (1 μM) and/or MEHP (200 μM) for our subsequent experiments in TM4 cells (Fig. 4A-B). After exposure to MEHP, the results of KEGG enrichment analysis in TM4 cells also showed the enrichment of various pathways, including AJ, GJ and TJ (Fig. 4C). The BTB is constituted by AJ Sertoli cells and composed of coexisting TJ, and desmosome–GJ. Thereby the in vitro data were in consistent with in vivo results, DEHP-induced spermatogenic disorders might be caused by disruption of BTB between Sertoli cells. To further authenticate the impact of DEHP-induced BTB disruption on spermatogenesis, we next examined changes in the protein levels of cell junctions in TM4 cells. TM4 treated with MEHP showed decreased levels of AJ proteins (E-cadherin, N-cadherin, α-catenin, β-catenin, γ-catenin), GJ proteins (Cx43), TJ proteins (Claudin 5, ZO-1, Occludin) and Vimentin, but they were reversed by LYC treatment (Fig. 4D and S6A-B). Subsequently, IF detection was performed to further confirm this effect. The levels of E-cadherin, N-cadherin, Cx43, ZO-1, Occludin and Vimentin proteins were slightly lower in DEHP-treated TM4 cells with a significant difference and the locations of these proteins were not different (Fig. 4E and S7A). However, these proteins levels showed a slight increase after LYC administration. Then, we used scratch test to assess cell migration ability. Our data showed the migration capacity of TM4 cells was obviously suppressed after exposure to MEHP, but LYC treatment could partly rescue the reduced cell migration ability (Fig. 4F and S6C). The expression of cytoskeletal components (F-actin and a-tubulin) was also decreased and disrupted, but localization remained the same in TM4 cells. However, LYC could prevent DEHP-induced decrease and disorder of cytoskeletal components, suggesting that LYC protected TM4 cell against DEHP-induced cytoskeletal alteration and cell migration ability decline (Fig. 4G and S8A-B).

Fig. 4.

LYC prevented DEHP-induced disruption of continuous cell junctions in TM4 cells. (A) TM4 cells were incubated with Con (control), LYC (1 μM LYC), MEHP (200 μM MEHP) and ML200 (1 μM LYC + 200 μM MEHP), respectively. (B) The cell viability was assayed using CCK-8 after exposure to different concentrations of LYC in TM4 cells. (C) KEGG analysis based on RNA-seq of TM4 cells after exposure to M200. (D) The levels of AJ, TJ, GJ and Vimentin proteins in TM4 cells. (E) Expression and positioning of E-cadherin, N-cadherin, Cx43, Occludin, ZO-1 and Vimentin proteins in TM4 cells. (F) Changes of cellular migration ability in TM4 cells. (G) Changes of cytoskeleton organization in TM4 cells. Data are presented as the mean ± SD. Symbol for the significance of differences between the Con group and another group: *P < 0.05, **P < 0.01.

Primary SCs cultured in vitro can form a TJ barrier-like structure between cells under specific culture conditions. TER was analyzed by a cell resistance meter to simulate and evaluate TJ and barrier function in vitro [38]. TER is an exogenous classical method commonly used in the study of functional impairment of reproductive toxicants BTB. In order to analyze the BTB dynamics, primary culture of SCs from was used for mimicking typical BTB structures (Fig. S9A). TER analysis showed that MEHP inhibited the Sertoli cell barrier function compared with Con group, but LYC treatment alleviated this change (Fig. S9B). Taken together, these results show that LYC regulates MEHP-induced disruption of BTB in Sertoli cells.

LYC ameliorates DEHP-induced BTB disruption by regulating Sertoli cell Cx43 expression

Cx43 has a pivotal role in the formation of gap junctions. In addition, CX43 in Sertoli cells may be crucial for BTB structure and function [39] (Fig. 5B). In mammalian cells, the function of Cx43 is tightly controlled by its phosphorylation state [40]. Various compounds can affect the regulation of Cx43 expression. In our study, we found that LYC inhibited DEHP-induced Cx43 phosphorylation and decreased protein level of Cx43, indicating that Cx43 may play a role in reversing DEHP-induced BTB disruption (Fig. 1C and S2B). Therefore, we focused on Cx43 for further studies. To this end, TM4 and SC cells were transfected with a validated expression vectors or siRNA were utilized to overexpress or knock down Cx43 (Fig. 5A). Indeed, overexpression of Cx43 significantly rescued MEHP-induced decreased cell migration ability in TM4 cells and reduced TER in SCs cells. Consistently, Cx43 knockdown led to reduction of endogenous Cx43 protein levels, and significantly reduced cell migration ability in TM4 cells and TER in SCs cells (Fig. 5C-D and S9C-E). This result further demonstrates that the Cx43 is essential for the regulation of BTB integrity. Our results strongly argue for the crucial role of Cx43 in LYC against DEHP-induced BTB damage in mice.

Fig. 5.

LYC ameliorates DEHP-induced BTB disruption by regulating Sertoli cell Cx43 expression. (A) TM4 or SCs cells were incubated with NC + Con, Cx43 + Con, NC + M200, and Cx43 + M200, or siNC + Con, siCx43 + Con, siNC + M200, and siCx43 + M200. (B) LYC preventing DEHP-induced BTB disruption by regulating Cx43. (C) Changes of Cx43 levels and cellular migration ability in TM4 cells. (D) Changes of Cx43 levels and TER in SCs. Data are presented as the mean ± SD. Symbol for the significance of differences between the NC + CON or siNC + Con group and another group: *P < 0.05, ***P < 0.001. Symbol for the significance of differences between the NC + M200 or siNC + M200 group and Cx43 + M200 or siCx43 + M200 groups: ^##P < 0.01, ^###P < 0.001.

Discussion

Male infertility is a growing global health concern, with 50 % of infertility cases caused by male-factor infertility [41]. Although, Tremendous efforts have been implemented into improving the male infertility, many gaps remain in our understanding of the mechanisms regulating spermatogenesis process, including the role of BTB. The panel agreed that the toxicological evidence for phthalate-induced male infertility is strong [42]. It is important not only to treat phthalate-induced male reproductive diseases but also to screen effective antagonists. As the “Plant Gold”, LYC is protective of prostate cancer and help in improving sperm viability. We speculate that LYC supplement may become a therapeutic approach to treat DEHP-induced male infertility. Thus, we examined the functional contribution of LYC and/or DEHP in the testis through in vivo and in vitro culture studies using different testicular cells. Here, we uncovered a LYC-mediated complementary BTB repair pathway that reversed potentially spermatogenesis disturbance caused by DEHP. We envision that LYC improves BTB integrity, which creates a distinct microenvironment for spermatogenesis and sperm maturation, and thus alleviates DEHP-induced spermatogenesis disorder. Through overexpression and RNA interference approaches, we identify Cx43 as a target of LYC and DEHP in mediating BTB integrity. We recognize an undiscovered function of Cx43 as a molecular switch for maintaining BTB integrity and an antagonist of male infertility. Above all, the present study suggests that Cx43 activated by LYC may provide a promising male infertility therapy, a conclusion supported by the evidence of our study. Maintaining Cx43 level in testis is thus essential for treatment of major reproductive disease.

The BTB contributes to normal male fertility by preventing the production of antibodies against sperm and leading to male infertility, as well as limiting the entry of harmful compounds [43]. Therefore, the presence of the BTB poses a unique clinical challenge in the treatment of several diseases. The BTB may serve as a sanctuary, protecting germ cells from xenobiotic toxicant exposure [44]. Our result indicated that DEHP led to the change of integrity and permeability in BTB, but LYC repaired this damage. The BTB is composed of three types of cellular junctions including TJ, GJ and AJ. Defects in the cell junctional proteins that establish BTB can lead to BTB dysfunction, which may trigger an immune response against meiotic and postmitotic germ cells, eventually causing male infertility [45]. Moreover, lack in proteins that regulate cell junction formation and function can also damage BTB function. Indeed, we have showed that exposure to DEHP led to BTB-related proteins levels decreased. Of note, LYC can regulate and promote BTB-related proteins expression to inhibit DEHP-induced BTB disfunction. This upregulation of BTB-related proteins can maintain BTB integrity and inhibit harmful compounds entering into the lumen. We also found that intracellular localization of BTB-related proteins was not altered. Thus, we speculate that LYC suppresses the DEHP and its metabolite entry to seminiferous tubule lumen by activating the expression of BTB-related proteins.

Spermatogenesis is entirely dependent on the testicular BTB integrity. Consistently, our analysis indicated significant enrichment of genes associated with spermatid development, spermatid differentiation and spermatid differentiation, which is mediated by DEHP. Spermatogenesis is a complex developmental process that occurs in the seminiferous tubules and ultimately generates mature sperm cells [46]. The process of sperm production is from spermatogonia to mature spermatozoa. Our further investigations suggested that DEHP exposure caused many morphological abnormalities of the spermatogonia and elongated sperm. Consistently, DEHP exposure also resulted in abnormal structure of sperm, such as head deformities, folded tails and flagellar kinked. However, LYC treatment restored their normal morphology. Malformed spermatozoa are a common cause of male infertility. Male infertility results from defects in spermatogenesis up to diminished fertilization ability once the spermatozoa reach the egg [47]. Spermatozoa penetrating the cumulus cells analysis is used to predict the fertilizing capacity of sperm. In our study, DEHP inhibited the cumulus-penetrating abilities of sperm, and prevented the sperm from advancing and combined with egg. As expected, LYC accelerated the ability of sperm to dissociate cumulus cell layer and migrate to the eggs. It is a powerful mechanism that LYC prevents DEHP-induced sperm damage, deformity and eventually, male infertility. Overall, the results shows that targeted regulation of BTB function disorder is a tantalizing but unknown therapeutic way for new sperm to genesis disturbance treatment.

BTB is a crucial ultrastructure maintaining meiotic and postmeiotic sperm cell development. The adhesion of germ cells to Sertoli cells and Sertoli cells to Sertoli cells are involved with BTB structure [48]. MEHP is the active and most toxic metabolite of DEHP. The data showed that KEGG significantly involved pathways included TJ, GJ and AJ in TM4 cells. The vitro results were also in agreement with the result in vivo, MEHP significantly suppressed the levels of TJ, GJ and AJ proteins in GC-1cells and TM4 cells, and LYC reversed these changes. KEGG also significantly involved pathways included focal adhesion and regulation of actin cytoskeleton. The junctions between cells link cells to each other and regulate many crucial processes, especially barrier function and migration [49]. Because a pivotal characteristic of cell migration is that migratory cells maintain contact through diverse cellular connections, we aimed to confirm whether the effect of LYC and/or DEHP on cell migration. Consistently, we found that LYC reversed MEHP-mediated inhibition of cellular migration of GC-1 cells and TM4 cells. Cell migration is driven by the cytoskeleton especially the constant crosstalk between microfilaments and microtubules [50]. During migration, cells form new adhesions and mature into focal adhesions, transmitting the traction forces involved in movement. In the present study, we showed that LYC could regulate the two cytoskeletal structures (actin microfilaments and microtubules) to disinhibit DEHP-induced decrease and disorder of cytoskeleton levels. The cytoskeleton plays a key role in maintaining BTB integrity in the testis, especially the cytoskeletal elements of AJ, which has been proposed as a vehicle for germ cells transport [51]. Consistently, we showed that LYC alleviated MEHP-induced damage to the BTB function in SCs. Other significantly involved pathways included lysosome, Parkinson disease, phagosome, protein processing in endoplasmic reticulum, mitophagy, autophagy, HIF-1 signaling pathway, chemical carcinogenesis - reactive oxygen species, and glutathione metabolism. In agreement with more recent results, mitophagy and antioxidant might be associated with the beneficial role of LYC in inhibiting DEHP-induced spermatogenic damage [52]. Furthermore, p53 signaling pathway, TNF signaling pathway, Cell cycle, MAPK signaling pathway, PI3K-Akt signaling pathway are also involved in KEGG pathways, which suggested more research opportunities that we will explore later, and we will further investigate this on future work. Therefore, these data suggest that LYC can prevent cytoskeletal disorders, decreased cell migration, and impaired barrier function caused by DEHP-induced the decrease of cell junction in testis.

CX43 plays a crucial role in the process of sperm production by mediating for direct cell communication between adjacent testicular cells [53]. This communication ensures Sertoli cells couple to germ cells through intercellular channels and allows the normal proliferation, differentiation and metabolism of male germ cells [54]. In this study, we revealed that DEHP promoted Cx43 phosphorylation and inhibited Cx43 protein level, but LYC reversed these changes in testicular tissue and its cells. Furthermore, several studies prove that Cx43 may be important for cell junction formation and remodeling [55]. It has been proven that Cx43 level is changed or reduced in men in the presence of impaired spermatogenesis. Specific knockout of Cx43 in Sertoli cells has an adverse effect on BTB dynamics and composition, and thus results in male mice infertile [39]. As expected, overexpression of Cx43 alleviated MEHP-induced the decline of cell migration ability in TM4 cells and the impairment of BTB function in SCs. Knockdown of Cx43 could inhibit the protective role of LYC on MEHP-induced the decline of cell migration ability in TM4 cells and the impairment of BTB function in SCs. It is suggested that LYC could antagonize DEHP-induced destruction of the BTB in mice by regulating Cx43.

Conclusion

In summary, we have suggested that LYC is highly protective in DEHP-perturbed Sertoli cell BTB integrity both in vitro and in vivo, affecting the TJ-permeability barrier function and cell migration capacity via changes in the homeostasis of cytoskeleton at the Sertoli–Sertoli and Sertoli–spermatid adhesion sites. We also identify the Cx43 as a key effector of BTB to promote spermatogenesis in testis. We further propose that LYC supplement targeting of Cx43 can be exploited to prevent DEHP-induced BTB disruption and thus treat spermatogenic disorders. Our study, therefore, provides ideas for developing therapeutic strategies for preventing male reproductive disease and identifies a potentially effective therapeutic strategy for treating phthalates-induced male infertility.

CRediT authorship contribution statement

Yi Zhao: Conceptualization, Investigation, Writing – original draft. Jia-Gen Cui and Hao Zhang: Methodology, Formal analysis. Jia-Xin Wang and Ming-Shan Chen: Visualization, Supervision. Xue-Nan Li: Conceptualization, Methodology, Writing – review & editing. Jin-Long Li: Writing – review & editing, Data curation, Methodology.

Compliance with ethics requirements

Ethics requirements for the trial was received from the Northeast Agricultural University Animal Care and Use Committee (No. NEAUEC20220341).

Competing Interest

The authors declare that they have no conflict of interest.

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.